Targeting platelet-leukocyte interactions:: Identification of the integrin Mac-1 binding site for the platelet counter receptor glycoprotein Ibα

The firm adhesion and transplatelet migration of leukocytes on vascular thrombus are dependent on the interaction of the leukocyte integrin Mac-1 (alpha(M)beta(2), CD11b/CD18) and the platelet counter receptor glycoprotein (GP) Ibalpha. Previous studies have established a central role for the I domain, a stretch of similar to200 amino acids within the am subunit, in the binding of GP Ibalpha. This study was undertaken to establish the molecular basis of GP Ibalpha recognition by alpha(M)beta(2). The p(201)-K-217 sequence, which spans an exposed loop and amphipathic alpha4 helix in the three-dimensional structure of the alpha(M)I domain, was identified as the binding site for GP Ibalpha. Mutant cell lines in which the alpha(M)I domain segments p(201)-G(207) and R-208-K-217 were switched to the homologous, but non-GP Ibalpha binding, alpha(L) domain segments failed to support adhesion to GP Ibalpha. Mutation of amino acid residues within P-201-K-217, H(210)A(212), T-213-I-215, and R-216-K-217 resulted in the loss of the binding function of the recombinant alpha(M)I domains to GP Ibalpha. Synthetic peptides duplicating the P-201-K-217, but not scrambled versions, directly bound GP Ibalpha and inhibited alpha(M)beta(2)-dependent adhesion to GP Ibalpha and adherent platelets. Finally, grafting critical amino acids within the P-201-K-217 sequence onto alpha(L), converted alpha(L)beta(2) into a GP Ibalpha binding integrin. Thus, the P-201-K-217 sequence within the alpha(M)I domain is necessary and sufficient for GP Ibalpha binding. These observations provide a molecular target for disrupting leukocyte-platelet complexes that promote vascular inflammation in thrombosis, atherosclerosis, and angioplasty-related restenosis.